Aberrant imprinting in humans: Application of genomewide approaches to better understand imprinting regulation and its disturbances

印记障碍(ImpDis)包括先天性疾病，其特征是父母印记基因的紊乱。这些基因的表达来自父亲或母亲的等位基因，这些干扰包括基因组和表观遗传学的改变。ImpDis的临床诊断经常因缺乏临床特异性而受到阻碍，因为症状可能会重叠。ImpDis是由四种不同的分子变化引起的：其中三种代表经典的分子变化，但表观突变包括DNA修饰(例如5-甲基胞嘧啶)。在大多数病例中，表观突变的原因尚不清楚，但印迹区域本身的分子变化(顺式因子)以及其他基因组区域的分子变化(反式因子)可能起了作用。然而，由于技术限制以及ImpDis的稀有性，目前缺乏系统的方法。随着NextGenerationSequence和Long-ad测序技术的实施，现在可以用合适的分析方法来全面分析表观遗传调节和IST干扰的基因组、结构、表观遗传和转录因素。这项建议将第二代和第三代测序方法与具有11p15.5和14q32表观突变的独特患者队列相结合，以解决蛋白质和非蛋白质编码区等位基因分辨率的基因组和表观遗传变异。确定ImpDis分子基础的策略包括：(A)全基因组分析11p15.5和14q32表位突变携带者的甲基化，以识别多位点印迹干扰(MLID)并区分ImpDis亚群。(B)鉴定引起mlid和孤立上突变的顺式和反式因子中的基因组变异。(C)一次在远距离覆盖整个差异甲基化区域，在单分子水平上确定特定等位基因甲基化模式。(D)确定大的结构变体的特征，以确定新的调节元件。总之，我们的目标是确定调节基因组印迹和印迹基因表达的因子和功能基因组区域。我们将在相同的分析中确定异常甲基化的表观全基因组模式以及染色体区域和参与印记调控的因素。分析不会局限于特定的基因组区域，但该提议将涵盖患者及其父母的整个基因组。结合转录组数据，可以描述干扰印迹的功能后果。这种方法将有助于理解ImpDis的病因学，但它也将被翻译用于患者的临床管理。

Imprinting disorders (ImpDis) comprise congenital diseases which are characterised by disturbances of parentally imprinted genes. These genes are expressed either from the paternal or the maternal allele, and the disturbances consists of both genomic and epigenetic alterations. The clinical diagnosis of ImpDis is often hampered by the lack of clinical specifity, as the symptoms can overlap. ImpDis are caused by four different molecular changes: Three of them represent classical molecular changes, but epimutations comprise DNA modifications (e.g. 5-methylcytosine). The causes of epimutations are unknown in the majority of cases, but molecular alterations in imprinted regions themselves (cis factors) as well as in other genomic regions (trans factors) probably play a role. However, systematic approaches are currently missing, due to technical limitations as well as the rareness of ImpDis. With the implementation of NextGenerationSequencing and Longread-Sequencing techniques suitable assays are now available fort he comprehensive analyses of genomic, structural, epigenetic and transcriptional factors of epigenetic regulation and ist disturbances. This proposal combines second and third generation sequencing approaches with a unique cohort of patients with 11p15.5 and 14q32 epimutations to address genomic and epigenetic variations at allelic resolution in protein and nonprotein coding regions. The strategy to identify the molecular basis of ImpDis includes (a) Genome-wide analysis of methylation in carriers of 11p15.5 and 14q32 epimutations to identify multilocus imprinting disturbances (MLID) and to discriminate ImpDis subgroups. (b) Identification of genomic variants in cis and trans factors causing both MLID and isolated epimutations. (c) Determination of allele-specific methylation patterns on single molecule level over long distance covering whole differentially methylated regions at once. (d) Characterization of large structural variants to identify new regulating elements. Altogether, we aim to identify factors and functional genomic regions which regulate genomic imprinting and the expression of imprinted genes. We will both determine epigenomewide patterns of aberrant methylation as well as chromosomal regions and factors involved in the regulation of imprinting in the same assays. The analysis will not be restricted to specific genomic regions but the proposal will cover whole genomes of both the patients and their parents. In combination with transcriptome data functional consequences of disturbed imprinting can be delineated. This approach will help to understand the pathoetiology of ImpDis, but it will also be translationally used for clinical management of the patients.